Methods of modulating tissue growth and regeneration

ABSTRACT

Polyionic derivatives of cyclodextrins and methods for preparing these derivatives are provided in which a polyionic derivative of cyclodextrin is combined with a growth factor, preferably a heparin binding growth factor. These compositions are of low solubility and are applied directly to the location of a wound. By virtue of the low solubility, the compositions remain in place at the site of application and slowly release growth factor. In an alternative embodiment, the cyclodextrin derivatives are administered in the absence of growth factor and are used to absorb growth factor present in the body at the location of the wound in order to prevent overstimulation of the wound response.

This is a division of application Ser. No. 08/345,011, filed Nov. 23,1994, now U.S. Pat. No. 5,658,894, which is a continuation ofapplication Ser. No. 07/900,592, filed Jun. 18, 1992 (abandoned) and acontinuation-in-part of application Ser. No. 07/790,320, filed Nov. 12,1991 (abandoned), which is a continuation of application Ser. No.07/691,168, filed Apr. 24, 1991 (abandoned), which is a continuation ofapplication Ser. No. 07/397,559, filed Aug. 23, 1989 (abandoned).Application Ser. No. 07/900,592 is a continuation-in-part of applicationSer. No. 07/480,407, filed Feb. 15, 1990 (U.S. Pat. No. 5,183,809,issued Feb. 2, 1993). All of the aforesaid applications and issuedpatent are hereby incorporated by reference.

FIELD OF INVENTION

The present invention is directed to compounds, compositions and methodsfor healing wounded living tissue, and particularly to saccharide-basedcompounds and compositions which remain localized at the site of a woundfor extended periods of time.

BACKGROUND OF THE INVENTION

The injury of tissue initiates a series of events that result in tissuerepair and healing of the wound. During the first several days followingan injury, there is directed migration of neutrophils, macrophages andfibroblasts to the site of the wound. The macrophages and fibroblastswhich migrate to the wound site are activated, thereby resulting inendogenous growth factor production, synthesis of a provisionalextracellular matrix, proliferation of fibroblasts and collagensynthesis. Finally from about two weeks to one year after infliction ofthe wound there is remodeling of the wound with active collagen turnover and cross linking (Pierce et al., 1991, J. Cell Biochem.,45:319-326). The manner in which this repair process is regulated ismostly unknown; it is known, however, that cell proliferation, migrationand protein synthesis can be stimulated by growth factors that act oncells having receptors for these growth factors.

In vivo studies have shown that local application of exogenous singlegrowth factors or a combination of growth factors can enhance thehealing process following experimental wounding in animals (Antoniadeset al., 1991, Proc. Natl. Acad. Sci. USA, 88:565-569). The ability ofthese growth factors to promote wound healing has resulted in efforts toobtain these factors in purified form. It is known that a number ofthese growth factors, known as heparin binding growth factors (HBGFs),have a strong affinity for heparin (reviewed in Lobb, 1988, Eur. J.Clin. Invest. 18:321-328 and Folkman and Klagsbrun, 1987, Science235:442-447). Accordingly heparin affinity chromatography has been usedto obtain these growth factors in purified form. In addition the DNAcoding for a number of these growth factors has been isolated and theproteins can be produced by recombinant DNA methods. These HBGFs havebeen shown to have mitogenic and non-mitogenic effects on virtually allmesoderm and neuroectoderm derived cells in vitro. HBFGs are also knownto promote the migration, proliferation and differentiation of thesecells in vivo. It was suggested by Lobb (1988, Eur. J. Clin. Invest.,18:321-328) that HBGFs could therefore effect the repair of soft tissue.It was further suggested that HBGFs may be used to effect the repair ofhard tissue such as bone and cartilage. In contrast to their beneficialeffects, it is also known that growth factors may over-stimulate thewound healing response, resulting in the excessive smooth muscle cellproliferation and migration which occur, for example, in restenosisfollowing angioplasty.

Knowledge of the affinity of growth factors for heparin and thedifficulty of obtaining heparin in a pure, homogeneous form has resultedin attempts to obtain a compound which possesses heparin's affinity forgrowth factors but which could be easily and reproducibly manufactured.As described in the parent applications referenced hereinabove, onegroup of compounds meeting these requirements are cyclodextrins, cyclicoligosaccharides consisting of up to at least six glucopyranose units.

U.S. Pat. No. 5,019,562 to Folkman et al. (the Folkman et al. patent),which is in the lineage leading to the present application, is directedto the use of highly soluble cyclodextrin derivatives to treatundesirable cell or tissue growth. The cyclodextrin derivativesdisclosed in this patent are combined with growth inhibiting steroids oradministered alone to absorb growth factors present in the blood stream.The cyclodextrin derivatives disclosed in the Folkman et al. patent arehighly hydrophilic and therefore highly soluble. The high solubility ofthese derivatives is said to be an important factor which cooperativelyinteracts with the inherent complexing ability of the cyclodextrinstructure for exogenous steroids. In addition, the high solubility ofthese compounds is said to facilitate introduction of the compounds intothe body and to aid in dispersal via the blood stream.

SUMMARY OF THE INVENTION

The high solubility of the compounds disclosed in the Folkman et al.patent is desirable for systemic administration of these compositions tothe body; on the other hand, however, applicants have found that thehigh solubility of these compounds limits their ability to remainlocalized in the area of a wound following administration. To maximizedelivery of a given growth factor or factors to a wound site, applicantshave discovered that it is desirable to obtain saccharide-basedcompounds possessing a high affinity for growth factor and very lowsolubility. According to one aspect of applicants' discovery, such lowsolubility compounds are combined with a growth factor prior toadministration to the body and applied locally to the site of a wound.Due, at least in part, to their low solubility, such compounds remain atthe site of application and slowly release the growth factor to optimizethe dosage of growth factor at the wound site. Applicants have alsofound that, alternatively, a compound possessing both a high affinityfor growth factor and a low solubility can be used to remain at the siteof an injury and to absorb at least some portion of the growth factorsreleased by the injured tissue, thereby reducing the probability ofover-stimulation of the wound healing process, as is observed inrestenosis following angioplasty.

In view of both the beneficial and pathological properties of growthfactors involved in wound repair, applicants have thus identified a needfor compositions which regulate the concentration and/or diffusion ofgrowth factors in the area of a wound so as to optimize the woundhealing process. Accordingly, the present invention provides lowsolubility polyanionic saccharide derivatives having a high negativecharge density for affecting the growth of living tissue in mammals.Also provided are compositions comprising an active agent comprising lowsolubility polyanionic saccharide derivative and a physiologicallyacceptable carrier for the saccharide derivative.

The saccharide derivative preferably has a body temperature solubilityof less than about 15 grams per 100 ml of water. According to certainpreferred embodiments, the saccharide derivatives have substantially nosolubility in water at body temperature. The term "body temperature" asused herein refers to the range of body temperatures expected for aliving mammal, including the lowered body temperatures used in varioussurgical techniques and the elevated body temperatures encountered inphysiological responses to infection. Unless otherwise indicated,solubility refers to solubility in distilled water.

The compositions of the present invention offer a number of advantagesover prior art compositions due, at least in part, to the low solubilityof the active ingredient in body tissues and fluid. The low solubilityof the present saccharide derivatives is advantageous in wound healingmethods which provide for administration directly to the site of awound. The compositions remain substantially at the administeredlocation for an extended period of time. When combined with growthfactors, the saccharide derivatives of the present invention facilitatecontrolled release of the growth factors at the wound site, therebyregulating and greatly enhancing the wound healing process. In theabsence of a growth factor, the present compositions can, by virtue oftheir affinity for growth factors, reduce the local concentration and/ordiffusion of growth factors produced by cells at the wound site as wellas growth factors present in the blood stream. By reducing the diffusionof growth factors, the compositions are capable of preventing orsubstantially reducing over-stimulation of the wound healing response,thereby avoiding the pathological growth of cells that results in suchconditions as restenosis following angioplasty, vein graft intimalhyperplasia, and native vessel atherosclerosis.

The present invention also provides methods for the preparation ofbeneficial wound healing compositions. These compositions compriserelatively insoluble solid forms of highly anionic polysaccharides. Themethod aspects comprise reacting a saccharide with an anionicderivatizing agent to generate a polyanionic derivative of thesaccharide, followed by salt formation of the largely insoluble product.Alternatively, saccharides are reacted with a suitable coupling agent togenerate a sparsely soluble polymer or copolymer of that saccharide,followed by a reaction with an anionic derivatizing agent. According tocertain embodiments, these derivatized saccharides are then combinedwith one or more growth factors. The compositions provided by thesemethods of preparation have the advantageous properties of very lowsolubility and high growth factor affinity.

The present invention also provides wound healing methods. According tothese methods, the present low solubility, polyanionic saccharidederivatives are applied to the area to be treated. Such methods areadaptable for use in the prevention of restenosis, promotion ofangiogenesis, treatment of transplanted tissue or organs and treatmentof damaged or transplanted bone or cartilage.

The ability of the present compounds and compositions to regulate thewound healing process offers possible life-saving benefits to patientswho have undergone procedures such as percutaneous transluminalangioplasty (hereinafter "PCTA"). It has been observed that up to 40% ofpatients who undergo PCTA are afflicted by restenosis and the recurrentarterial blockage that it causes. Thus, the long-term effectiveness oftreatments for arteriosclerosis, such as angioplasty, have beensubstantially limited by the reoccurrence of restenosis. It is believedthat the present compositions will substantially reduce or eliminaterestenosis and thereby have a major influence on the morbidity andmortality rate for patients which have undergone angioplasty, vein graftbypass operations and similar procedures. In addition, it is expectedthat victims of cardiac, cerebral or peripheral ischemic disease willgreatly benefit from use of the compositions of the present invention.In particular, patients who suffer from infarcted myocardial tissuerequire the establishment of new collateral blood capillaries andvessels to supply blood to the infarcted tissue. The presentcompositions may include growth factors to promote angiogenesis at thesite of the infarcted tissue. These examples represent just a few of thepossible life-saving benefits offered by the compositions and methods ofthe present invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(A and B) is a schematic representation of (A) the chemicalstructure of α-, β- and γ-cyclodextrin monomer; and (B) of thethree-dimensional shape of these cyclodextrin monomers.

FIG. 2 is a schematic representation of the chemical structure ofsucrose with the sites of anionic substituent groups indicated.

FIG. 3 shows the affinity of beta-cyclodextrin tetradecasulfate polymerfor basic fibroblast growth factor.

FIG. 4 shows polyacrylamide gel electrophoresis of basic fibroblastgrowth factor and Chondrosarcoma derived growth factor purified bycyclodextrin copper biaffinity chromatography. Lane 1 shows the proteinprofile of the protein markers (phosphorylase b, bovine serum albumin,ovalbumin, carbonic anhydrase,soybean trypsin inhibitor, betalactoglobulin, and lysozyme). Lanes 2 and 3 show the 18,000 molecularweight polypeptide bands of basic fibroblast growth factor andChondrosarcoma derived growth factor, respectively.

FIG. 5 compares the affinities of heparin and beta-cyclodextrintetradecasulfate polymer for Chondrosarcoma derived growth factor.

DETAILED DESCRIPTION OF THE INVENTION

The invention is directed to compounds, compositions and methods foraffecting the growth of living tissue in mammals. The novel compounds ofthe present invention are derivatized cyclodextrin polymers having lowsolubility in distilled water at body temperature and a high negativecharge density. The present compositions comprise a low solubility,polyanionic saccharide derivative having a relatively high density ofanionic substituents and a carrier for such derivative.

One important aspect of the present compounds and compositions is thestrong affinity of such materials for proteinic growth factors. Althoughapplicants do not intend to necessarily be bound by or limited to anyparticular theory, it is thought that the density of the anionic groupson the saccharide compounds of the present invention is important inproviding the high affinity of these compounds for tissue and growthfactors. Applicants have discovered that the affinity of the presentcompounds and compositions for growth factors combined with the lowsolubility of the present saccharide derivatives provides the ability toregulate and control the concentration of growth factors in the area ofa wound. In addition, the present compounds and compositions provideactive agents in the form of the present derivatized saccharides whichtend to adhere to living tissue. As a result, such compositions andcompounds have the highly desirable ability to provide active woundhealing agents at the site of an injury for extended periods of time.

The invention is also directed to methods for preparing thesecompositions and to methods for treating a variety of wounds resultingfrom accidents or surgical procedures. As the term is used herein,"wound healing" refers; to the repair or reconstruction of cellulartissue. The wound may be the result of accident, such as injury orburns. The wounds treatable by the present compositions and methods alsoinclude wounds resulting from surgical procedures of any type, fromminor intrusive procedures, such as catheterization or angioplastyresulting in wounding of vascular or organ surfaces, to major surgicalprocedures, such as bypass or organ transplant operations. Included inthis concept of wound healing is the repair of injured or fragmentedbone or cartilage and the promotion of the establishment of bone graftsor implants.

I. THE COMPOSITIONS

Applicants have found that compositions comprising as; an active agentpolyanionic saccharide derivatives having a high negative charge densityand low solubility can be useful wound healing materials. Especiallypreferred are polyanionic cyclodextrin polymers.

As used herein, the term "polyanionic saccharide derivative" refersbroadly to saccharide based compounds having 1.3 or more anionicsubstituents per sugar unit. The term sugar unit as used herein refersto an elementary monosaccharide building block which may, for example,be a hexose or pentose. Exemplary monosaccharides are glucose, fructose,amylose, etc. It is contemplated that all compounds which include abasic saccharide structure, as well as homologues, analogues and isomersof such compounds, are within the scope of the term "saccharide" as usedherein. The saccharide compounds hereof may comprise, for example,disaccharides, trisaccharides, tetrasaccharides, oligosaccharides,polysaccharides and polymers of such saccharides. The term"oligosaccharide" refers to saccharides of from about 5 to about 10sugar units having molecular weights, when unsubstituted, from about 650to about 1300. The term "polysaccharide" refers to saccharidescomprising greater than about 10 sugar units per molecule.Polysaccharides are understood to be saccharides having many sugar unitspossessing a variety of structures and various substituent groups. Theterm polymer as used herein refers to structures of repeated and similarsaccharide compounds, based on monomers which are linked together toform the polymer.

Applicants have found that the relationship between the structure of thederivatized saccharide and the level of negative charge density caninfluence the effectiveness of the present compounds, compositions andmethods. For example, the anionic substituents are preferably present inthe molecule to an extent of from, on average, about 1.0 to about 4substituents per sugar unit. Especially preferred compounds are thosebased on saccharides having on average at least about 1.4 anionicsubstituents per sugar unit. For saccharide compounds comprised of nsugar units and R substituents, it is preferred that the anionicsubstituents on the derivatized saccharide correspond substantially toabout the following:

If

n=2 to 3; average anionic R per n unit=or >3.5

n=4 to 5; average anionic R per n unit=or >2.0

n=>6; average anionic R per n unit=or >1.4.

While applicants contemplate that the anionic substituents of thepresent invention may be selected from a large group of known andavailable anionic substituents, it is generally preferred that theanionic substituents be selected from the group consisting of sulfate,carboxylate, phosphate, sulfonate, and combinations of two or more ofthese. Preferred compositions are based on saccharides having 6 or moresugar units and from about 2 to about 3 substituents per sugar unit,wherein the substituents comprise sulfate, sulfonate and/or phosphatesubstituents.

The saccharide derivatives of the present invention have a lowsolubility in distilled water at body temperature. As the term is usedherein, "low solubility" refers to solubility of much less than about 15grams per 100 ml of water. It refers to the ability of the presentsaccharide compounds to remain localized in a solid state for asubstantial length of time in an aqueous medium such as physiological ordistilled water. According to certain preferred embodiments, thesaccharide derivatives have substantially no solubility in distilledwater at body temperature. That is, it is preferred that the solubilityof the saccharide derivative is much less than about 1 gram per 100 mlof distilled water, and even more preferably less than about 1 milligramper 100 ml. Such insolubility is achieved, for example, by utilizingsaccharide compositions comprising polymer aggregates or dispersions ofsubstantially solid polymer particles. While it is contemplated thatvarious particle sizes and shapes may be utilized, it is preferred thatthe particles have an average particle size ranging from about amillimicron to about 1000 microns in diameter. Expressed in terms ofmolecular weight, the polymers comprising the polymer have, on average,a molecular weight of about one billion or greater. The high molecularweight of the preferred polymers is due to the presence of many millionsof sugar units within any of the discrete undissolved entities.Alternatively, in other embodiments of the invention, particles havingthe desired insolubility are produced by forming a salt comprising ananionic saccharide in combination or associated with a polyvalentcationic constituent.

While the compositions of the present invention may be produced from thesoluble saccharides as starting materials, as indicated above, it isalso possible to employ as starting materials a sparsely soluble, quasisolid or solid saccharides, such as cellulose or starch. Utilization ofthese saccharide sources preferably comprises chemically orenzymatically degrading the solid saccharide, followed by providing thesubstituent groups in accordance with this invention.

A. Cyclodextrin Derivatives

Especially preferred according to the present invention are compositionscontaining a cyclodextrin derivative. Cyclodextrins are saccharidecompounds containing at least six glucopyranose units forming a ring ortoroid shaped molecule, which therefore has no end groups. Althoughcyclodextrins with up to twelve glucopyranose units are known, only thefirst three homologs have been studied extensively. These compounds havethe simple, well-defined chemical structure shown in FIG. 1(A). Thecommon designations of the lower molecular weight α-, β- andγ-cyclodextrins are used throughout this specification and will refer tothe chemical structure shown in FIG. 1(A) wherein n=6, 7, or 8glucopyranose units, respectively. The initial discovery of thecyclodextrins as degradation products of starch was made at about theturn of the century, and Schardinger showed that these compounds couldbe prepared by the action of Bacillus macerans amylase upon starch. Inolder literature, the compounds are often referred to as Schardingerdextrins. They are also sometimes called cycloamyloses.

Topographically, the cyclodextrins may be represented as a torus, asshown in FIG. 1(B), the upper rim of which is lined with primary --CH₂OH groups, and the lower rim with secondary hydroxyl groups. Coaxiallyaligned with the torus is a channel-like cavity of about 5, 6 or 7.5A.U. diameter for the α-, β-, and γ-cyclodextrins, respectively. Thesecavities make the cyclodextrins capable of forming inclusion compoundswith hydrophobic guest molecules of suitable diameters.

The compositions of the present invention preferably include polyanioniccyclodextrin derivatives. In general, the terms "derivatized CD," "CDderivative" and the like refer to chemically modified CDs formed byreaction of the primary or secondary hydroxyl groups attached to carbons2, 3 or 6 of the CD molecule without disturbing the α (1→4) hemiacetallinkages. A review of such preparations is given in "Tetrahedron ReportNumber 147, Synthesis of Chemically Modified Cyclodextrins," A. P. Croftand R. A. Bartsch, Tetrahedron 39(9):1417-1474 (1983), incorporatedherein by reference in the background (hereinafter referred to as"Tetrahedron Report No. 147").

The CD derivatives are preferably derivatized cyclodextrin monomers,diners, trimers, polymers or mixtures of these. In general, thecyclodextrin derivatives of the present invention are comprised of orformed from derivatized cyclodextrin monomeric units consisting of atleast six glucopyranose units having α (1→4) hemiacetal linkages. Thepreferred derivatized cyclodextrin monomers of the present inventiongenerally have the formula (I): ##STR1## wherein at least two of said Rgroups per monomeric unit are anionic substituents and the remainder ofsaid R groups, when present, are nonanionic groups selected from wellknown and available substituent groups. The remaining, nonanionic Rgroups may be, for example, H, alkyl, aryl, ester, ether, thioester,thioether and --COOH. Exemplary alkyl groups include methyl, ethyl,propyl and butyl. The remaining nonanionic R groups may be hydrophilic,hydrophobic or a combination thereof, depending upon the particularrequirements of the desired composition. However, it is generallypreferred that the remaining nonionic R substituents be hydrophobic inorder to minimize the solubility of the compounds.

For CD monomers having the structure of Formula I wherein n is fromabout 6 to about 8, it is preferred that the compound have on average atleast about 9 anionic R substituents per monomer unit, more preferablyat least about 12 anionic R substituents per monomer, and even morepreferably at least about 14 anionic R substituents per monomer. Ingeneral it is preferred that the anionic substituents be relativelyevenly distributed on the monomer molecule, and accordingly compoundshaving the structure of Formula I wherein n is from about 6 to about 8preferably have from about 1 to about 3 anionic R substituents per nunit, more preferably from about 1.3 to about 2.5 anionic R substituentsper n unit and even more preferably from about 1.4 to about 2.2 anionicR substituents per n unit. Such structures are believed to provide thehigh negative charge density found to be therapeutically beneficial,with the highest charge density molecules providing excellent results.

The polyanionic cyclodextrin monomers of the type described above areimportant components of the preferred compositions of the presentinvention. The monomeric units may be present in the composition in theform of, for example, insoluble polymeric or co-polymeric structures oras insoluble precipitated salts of derivatized cyclodextrin monomer,dimer or trimer. Such salts may be formed by methods which comprisederivatizing the CD with anionic substituent and then complexing orassociating the derivatized CD with an appropriate polyvalent cation toform an insoluble derivatized CD salt. In alternative and preferredembodiments, the basic monomeric structure identified above comprisesthe repeating unit of novel insoluble polymeric cyclodextrins, asdescribed more fully hereinafter.

1. Cyclodextrin Polymers

According to important and preferred embodiments, the presentcompositions comprise derivatized cyclodextrin polymers. The presentpolymers have a structure corresponding to polymers formed fromderivatized cyclodextrin monomers of the type illustrated above. In viewof the present disclosure, it will be appreciated that polymericmaterials having such structure may be formed by a variety of methods.For example, derivatized cyclodextrin polymers may be produced bypolymerizing and/or cross-linking one or more derivatized cyclodextrinsmonomers, dimers, trimers, etc. with polymerizing agents, e.g.epichlorhydrin, diisocynanates, diepoxides and silanes using proceduresknown in the art to form a cyclodextrin polymer. (Insoluble CyclodextrinPolymer Beads, Chem. Abstr. No. 222444m, 102:94; Zsadon and Fenyvesi,1st. Int. Symp. on Cyclodextrins, J. Szejtli, ed., D. Reidel PublishingCo., Boston, pp. 327-336; Fenyvesi et al., 1979, Ann. Univ. Budapest,Section Chim. 15:13 22; and Wiedenhof et al., 1969, Die Starke21:119-123). These polymerizing agents are capable of reacting with theprimary and secondary hydroxy groups on carbons 6, 2, and 3.Alternatively and preferably, the derivatized cyclodextrin polymers maybe produced by first polymerizing and/or cross-linking one or moreunderivatized cyclodextrin monomers, dimers, trimers, etc. (eg.,cyclodextrins having the structure of FIG. 1) and then derivatizing theresulting polymer with anionic substituents. Underivatized cyclodextrinpolymer is available from American Maize Products Co., Hammond, Ind. inthe form of an epichlorhydrin linked polymer of β-cyclodextrin.Underivatized commercially available polymers may be derivatized toproduce the desired form of derivatized cyclodextrin polymer. Thederivatized cyclodextrin polymers may also be formed by reactingmixtures of derivatized monomers and underivatized monomers, or bycopolymerizing and/or crosslinking derivatized cyclodextrin polymers andunderivatized cyclodextrin polymers. For all preparation procedures, itis preferred that the polymerization method employed result in a solidpolymer product of sufficient porosity to allow diffusion penetration ofmolecules between the external solvent and a substantial portion of theinternal anionic monomer sites.

The solubility of the present CD polymers will depend, inter alia, onthe molecular weight and size of the polymer. The present derivatized CDpolymers are of large molecular weight so as to remain substantially inthe solid state. They are solid particulates of generally about 1 to 300micron size.

The derivatized cyclodextrin polymer of the present invention may beavailable in a variety of physical forms, and all such forms are withinthe scope of the present invention. Suitable forms include beads,fibers, resins or films. Many such polymers have the ability to swell inwater. The characteristics of the polymeric product, chemicalcomposition, swelling and particle size distribution are controlled,atleast in part, by varying the conditions of preparation.

The cyclodextrin polymer derivative preferably comprises a polyanionicderivative of an alpha-, beta-, or gamma-cyclodextrin polymer. Inpreferred embodiments the anionic substituents are selected from thegroup consisting of sulfate, sulfonate, phosphate and combinations oftwo or more of the foregoing. Although it is possible that other anionicgroups such as nitrate might possess some therapeutic capacity, thesulfate, sulfonate and phosphate derivatives are expected to possess thehighest therapeutic potential. In preferred embodiments, at least about10 molar percent of the anionic substituents, and even more preferablyat least about 50 molar percent, are sulfate groups. Highly preferredare alpha-, beta-, and gamma-cyclodextrin polymers containing about10-16 sulfate groups per cyclodextrin monomer, with beta-cyclodextrintetradecasulfate polymer being especially preferred.

B. Insoluble Salt Precipitates

The present compositions may include derivatized insoluble saccharidesalt precipitates, and preferably derivatized insoluble oligosaccharidesalt precipitates. As the term is used herein, "salt precipitate" meansa polyanionic saccharide derivative which has been associated orcomplexed with a suitable, non-toxic, physiologically acceptable cationto produce a salt which is substantially insoluble at body temperature.Suitable polyvalent cations which may be used to produce an insolublesalt precipitate of the present invention include Mg, Al, Ca, La, Ce,Pb, and Ba. The cations herein listed are presented generally in orderof decreasing solubility, although this order may be different forsaccharides of different types and degrees of anionic substitution.While all such derivatized insoluble saccharide salt precipitates arebelieved to be operable within the scope of the present invention, thederivatized oligosaccharides are preferred. Such oligosaccharidestypically have unsubstituted molecular weights ranging from about 650 toabout 1300. Oligosaccharides are usually obtained by procedures ofdegradation of starches or cellulose which result in oligosaccharidefragments in a broad range of sizes. Cyclodextrins are generallyobtained from starches in the presence of specific enzymes that favorthe formation of the cyclic saccharide structures. According to certainembodiments, the cyclodextrin salt precipitates are obtained by reactingthe desired cyclodextrin monomer or monomers with agents that willproduce the desired anionically substituted product and subsequentlyexchanging the cations which were introduced by the synthesis forcations of the desired polyvalent type. This latter step will result inprecipitation of the insoluble saccharide salt precipitate.

The Al, Ca and Ba salts of α-, β- and γ-CD sulfate are preferred for usein the compositions of the present invention, with Al β-CD sulfate saltsbeing preferred in certain embodiments. As with the saccharidederivatives generally, various degrees of sulfation per glucose unit canbe employed. It is generally preferred, however, that the derivatizedcyclodextrin salts have an average of at least about 1.3 sulfate groupsper sugar unit, and even more preferably about two sulfate groups persugar unit. Especially preferred is β-CD-TDS which has an average ofabout two sulfate groups per glucose unit.

C. Polyanionic Disaccharide Derivatives

Sucralfate (Carafate®, Marian Merrill Dow, Kansas City, Mo.) is acomplex salt of sucrose sulfate and aluminum hydroxide. Its structure isshown in FIG. 2. Sucralfate is an α-d-glucopyranoside,β-d-fructofuranosyl-, octakis(hydrogen sulfate) aluminum complex.Sucralfate is used to treat ulcers and was developed during studies ofsulfated polysaccharides that bind pepsins but lack anti-ulcer efficacy.The sulfation of sucrose and its conjugation with a basic aluminum saltresulted in a pepsin-inhibiting molecule suitable for treatment ofulcers. Denis M. McCarthy, Sucralfate, 325:14 New Eng. J. Med.,1017-1025 (1991).

Applicants have found that sucralfate and other polyionic derivatives ofsucrose have some properties in common with the derivatizedcyclodextrins of the present invention and may provide similarsolubility and affinity for growth factors. It is believed thatsulfonate or phosphate derivatives of sucrose combined with polyvalentcations such as Mg, Al, Ca, La, Ce, Pb or Ba may result in compositionsof low solubility which can be combined with growth factors tofacilitate therapeutic delivery of these growth factors to the site of awound. Oral administration of sucralfate has been described to havetherapeutic usefulness in the treatment of stomach ulcers. According tothe present invention, sucralfate and other salts of sucrose octasulfatemay be used to deliver growth factor proteins to tissues or bone in needof repair, by prior complexing with growth factors, and delivering thecomplex physically to the site of repair.

The frequent and/or high dosage use of aluminum salts is well known tohave certain health risks associated with it. Aluminum uptake is knownor suspected to be associated with a number of diseases. See, forexample, the extensive discussions in the books ALUMINUM AND HEALTH; ACRITICAL REVIEW (Hillel and Gitelman, Ed.), Mark Decker, Publisher, 1989and ALUMINUM IN RENAL FAILURE, Mark E. de Broi and Jack W. Coburn,Klewer, Publisher, 1990.

Aluminum is known to produce abnormalities in bone metabolism, such asosteodystrophy, osteomalacia, impaired mineralization, etc. Theintroduction of aluminum into the blood stream, such as can occur indialysis, can be particularly harmful. The following are but a fewexamples of reports concerning the harmful effects of aluminum: A. M.Pierides et al., Kidney Int., Vol. 18, 115-124, 1984; H. A. Ellis etal., J. Clin. Path. 32, 832, 1979. In addition to the toxic effects ofAluminum when introduced into the blood stream, oral administration ofaluminum salts can also produce a variety of harmful effects includingosteomalacia and osteitis; see, e.g., S. P. Andredi, J. M. Bergstein etal., N. Engl. J. Med., Vol. 310, 1079, 1984; K. A. Carmichael, M. D.,Fallon et al., Am. J. of Med., Vol. 76, 1137, 1984.

Particularly prominent among aluminum's toxic effects are neuralabnormalities, particularly Alzheimer's disease, in which aluminum issuspected to play an important role, although by a mechanism not yetunderstood. See, for example, D. R. Crapper McLachlan, B. J. Farnell,Aluminum in Neuronal Degeneration, in Metal Ions in Neurology andPsychiatry, pp. 69-87, 1985, Alan R. Liss Inc.; D. P. Perl, P. F. Good,Uptake of Aluminum into Central Nervous System Along Nasal-OlefactoryPathways, The Lancet, May 2, P. 1028, 1987; J. D. Birchall, J. S.Chappell, Aluminum, Chemical Physiology, and Alzheimer's Disease, TheLancet, October, P. 1008, 1988.

Given the possible toxicity of aluminum the non-aluminum salt forms ofthe highly sulfated polysaccharides are preferable over the aluminumsalts forms in some and perhaps all therapeutic applications. Inparticular, the polymeric embodiments which do not require saltprecipitate formation, are particularly preferred.

Several specific embodiments of the compositions of the presentinvention are particularly useful for oral administration in the healingof stomach ulcers. In particular, the non-aluminum salt-containing formsof sucrose octasulfate, and most preferably the polymeric solid form ofhighly sulfated cyclodextrin are especially advantageous because of theabsence of aluminum and its side effects.

D. The Form of the Compositions

In view of the disclosure contained herein, those skilled in the artwill appreciate that the present wound healing compositions are capableof having a beneficial effect in a variety of applications. It istherefore contemplated that the compositions of this invention may takenumerous and varied forms, depending upon the particular circumstance ofeach application. For example, the derivatized saccharide may beincorporated into a solid pill or may in the form of a liquid dispersionor suspension. In general, therefore, the compositions of the presentinvention preferably comprise a derivatized saccharide and a suitable,non-toxic, physiologically acceptable carrier for the saccharide. As theterm is used herein, carrier refers broadly to materials whichfacilitate administration or use of the present compositions for woundhealing. A variety of non-toxic physiologically acceptable carriers maybe used in forming these compositions, and it is generally preferredthat these compositions be of physiologic salinity.

For some applications involving would healing in the broadest sense, itis desirable to have available a physically applicable or implantablepredetermined solid form of material containing the therapeuticallyactive material of the invention. Accordingly, it is contemplated thatthe compositions of this invention may be incorporated in solid formssuch as rods, needles, or sheets. They may thus be introduced at or nearthe sites of tissue damage or sites of implantation, or appliedexternally as wound dressings, etc. In such embodiments, thecompositions and compounds of the present invention are preferablycombined with a solid carrier which itself is bio-acceptable, or thecompositions comprise suitably shaped polymer or co-polymer of thepresent saccharide derivatives. For many applications, it is preferredthat the compositions of the present invention are prepared in the formof an aqueous dispersion, suspension or paste which can be directlyapplied to the site of a wound. To prepare these compositions, apolyanionic saccharide derivative, such as polyanionic cyclodextrinpolymer, can be used as synthesized in solid form after suitablepurification, dilution and addition of other components, if desirable,including a fluid carrier, such as saline water. This will be the casewhen the product, saccharide salt, saccharide polymer or the saccharideco-polymer has been synthesized such as to produce a particle form ofprecipitate, dispersion or suspension. After synthesis, the solidderivative may also be dried, milled, or modified to a desired particlesize or solid form. The particle size can be optimized for the intendedtherapeutic use of the composition. In some preferred embodiments thesolid particles range in size from about 1 micron to about 600 microns,with from about 200-600 microns being even more preferred. Particlesranging from about 1 to about 30 microns offer the best dispersion ofgrowth factor and fast reactivity. For a given weight quantity ofparticles delivered to the biological environment, a smaller particlesize assures exposure of greater particle surface area allowing greaterdiffusion of proteinic active ingredients into or out of theadministered solid. Particles ranging from about 30 to about 100 micronsoffer fair dispersion of growth factors, medium reactivity and a longerperiod of delivery of growth factor. Particles possessing a size inexcess of 100 microns will have low reactivity, but provide the longestdelivery time for growth factors. In certain preferred embodiments,these large particles (>100 micron) will be used to absorb, rather thandeliver growth factors in vivo.

In preferred embodiments, the carrier is an aqueous medium and thecompositions are prepared in the form of an aqueous suspension of solidparticulate saccharide derivative. The amount of the derivatizedsaccharide preferably ranges from about 1 to 30% by weight of thecomposition, and even more preferably from about 5 to about 15% byweight.

E. Biologically Active Protein

In certain embodiments, the compositions and compounds include and/orare combined with biologically active proteins. According to preferredembodiments, the biologically active protein exhibits a specificaffinity for heparin, and, more specifically, is heparin-binding growthfactor, i.e., a class of growth factors, many of which are mitogenic forendothelial cells. An example of such a growth factor is basicfibroblast growth factor. Generally it will be the heparin-bindinggrowth factor proteins, commonly referred to as HBGF's, which may becombined with the saccharide derivatives of the present invention. Someof these are listed in Table I.

To determine whether a protein is suitable for the therapeuticcompositions of the present invention, one can determine whether it hasa specific affinity for heparin. A HBGF protein is one that remainssubstantially bound to heparin (e.g., using a derivatized column) evenin the presence of an aqueous medium having a salt concentration ofsubstantially greater than about 0.6 molar strength of NaCl. Generally,the term substantially bound refers to at least about 80% of such boundprotein remaining attached under such conditions.

                  TABLE I    ______________________________________    PROTEIN FACTORS    Symbol   Name         Reference    ______________________________________    IL-1     (Interleukin-1)                          Henderson & Pettipher, 1988,                          Biochem. Pharmacol. 37:4171;                          Endo et al., 1988, BBRC                          136:1007, Hopkins et al., 1988,                          Clin. Exp. Immunol. 72:422    IL-2     (Interleukin-2)                          Weil-Hillman et al., 1988, J.                          Biol. Response Mod. 7:424; Gemlo                          et al., 1988, Cancer Res.                          48:5864    IFNα             (Interferon α)                          Pitha et al. 1988, J. Immunol.                          141:3611; Mangini et al., 1988,                          Blood 72:1553    IFNγ             (Interferon γ)                          Blanchard & Djeu. 1988, J.                          Immunol. 141:4067; Cleveland et                          al., 1988, J. Immunol. 141:3823    TNFα             (Tumor necrosis                          Plate et al., 1988, Ann. NY             factor α)                          Acad. Sci. 532:149; Hopkins &                          Meager, 1988, Clin. Exp.                          Immunol. 73:88; Granger et al.,                          1983, J. Biol. Response Med.                          7:488    EGF      (Epidermal   Carpenter and Cohen, 1979, A.             growth factor)                          Rev. Biochem. 48:193-216    FGF      (Fibroblast  Folkman and Klagsbrun, 1987,             growth factor,                          Science 235:442-447             acidic and             basic)    IGF-1    (Insulin-like                          Blundell and Humbel, 1980,             growth factor-                          Nature 287:781-787; Schoenle et             1)           al., 1982, Nature 296:252-255    IGF-2    (Insulin-like                          Blundell and Humbel             growth factor-             2)    PDGF     (Platelet-   Ross et al., 1986, Cell 46:155-             derived growth                          169; Richardson et al.; 1988,             factor)      Cell 53:309-319    TGF-α             Transforming Derynck, 1988, Cell 54:593-595             growth factor-             α)    TGF-β             (Transforming                          Cheifetz et al., 1987, Cell             growth factor-                          48:409-416             β)    ______________________________________

It is known that the complexing capabilities of heparin toward growthfactor proteins are paralleled by its complexing capabilities forcertain cationic dye structures, such as azure-A, methylene blue andothers. Other glycosaminoglycan saccharides are known not to functionsimilarly. Thus such dyes have been used for many years in histology asspecific stains for the presence of heparin like polysaccharides; andmetachromasia, i.e. the spectral shift resulting from heparin binding onthe dye has been used to identify active heparin-like compounds havingthe capability of modulating angiogenesis. Such dye complexing of theactive protein also is similarly resistant to salt concentration as isthe complexing to heparin.

In relation to this invention it has been discovered that such dyecomplexing, serving as a model for proteinic growth factor complexing,can usefully serve as an indicator for the desired activity of thecompositions of the invention. Thus, the proteinic growth factorcomplexing ability of the precipitates, polymers, or co-polymers of thecompositions of the present invention may be determined using dyecomplexing assays.

In the course of practicing heparin binding separation or chromatographyfor the separation of proteinic factors it has been customary andaccepted that desorption of the complexed growth factor requires theadded step and involvement of contacting with a very strong saltsolution. The present invention makes use of the important discovery andrecognition that release of protein complexed to the saccharide hereinspecified does not require the added step of contacting with highconcentration electrolyte. While such operation would be needed for animmediate large scale desorption process as may be desired for aseparation technology, the relatively very low external concentration ofdesorbed factor is maintained by an equilibrium process involving thecomplexed phase on the solid and the low biologically required solutephase in the physiological surrounding liquid. This is a basic discoveryand recognition allowing the use of our compositions as delivery agentsfor biomedical purposes.

Generally, to prepare the growth factor containing compositions,derivatized saccharide is contacted with a solution containing a growthfactor or combination of growth factors. The cyclodextrin derivative isthereafter separated from the contact fluid, resulting in an enrichmentof the growth factor on the cyclodextrin derivative, and a correspondingremoval of the growth factor from the fluid. The contacting solution maycontain a single preseparated, preconcentrated growth factor purifiedfrom tissue or bodily fluids or growth factor obtained from recombinantDNA methods. Alternatively the contact solution may comprise viabletissue or organ materials (hereinafter organic sources) which contain avariety of growth factors. When combined with tissue or organ materialcontaining growth factors, the saccharide derivatives of the presentinvention may act as extractants of these growth factors. When organicsources are used as the source for growth factors, it is preferred thatthe organic source used for the contacting solution have a volumegreater than about 10 to about 100 times the volume of the tissue to betreated by the combined derivative and growth factor(s).

After contacting the partially or wholly completed saccharidederivative, the solid phase, can be easily separated from the fluidphase that was the source of protein to be complexed. It is preferablethat the source of growth factor contains the protein as a dissolvedcomponent in the absence of solids other than the saccharides to becomplexed. However, some solids in the growth factor source solution,may not necessarily be undesirable or disturbing contaminants.Separation of solids, such as tissue or organ fragments from thesaccharides, may be accomplished by sedimentation, suitable filtering,centrifugation or other mechanical or other methods.

II. METHODS FOR THERAPEUTIC REGULATION OF WOUND HEALING

One aspect of the present invention relates to methods for thetherapeutic regulation, and preferably in vivo regulation, of woundhealing, and particularly to in vivo regulation of the concentration anddiffusion of protein factors. Such methods generally comprisetherapeutic biodelivery of the present compositions and compounds to thewound site. The low solubility, i.e. the solid immobilized state, of thepresent materials allows the compositions and compounds to beadministered directly to the site of a wound and for the activeingredients to remain at the site of application for an extended periodof time.

Vascular cell proliferation and abnormal accumulation of extracellularmatrix in the vessel wall are common pathological features observed inarteriosclerosis, hypertension and diabetes. Such conditions are alsoobserved following vascular injuries, such as angioplasty. Intimalhyperplasia is thought to be mediated in part by a variety of growthfactors, such as platelet derived growth factor (PDGF), acting throughreceptors to stimulate vascular smooth muscle cell proliferation andmigration from the media into the intima. Thus, applicants havediscovered methods for regulating migration and proliferation of thesmooth muscle cells, thereby affecting the degree of intimal thickeningnoted after vascular injury. The applicants have found thatβ-cyclodextrin tetradecasulfate can inhibit human vascular smooth musclecell proliferation and migration in vitro when stimulated with fetalcalf serum, which contains potent growth factor activity.

It is seen, therefore, that the presence or absence of growth factors atthe site or vicinity of a wound has an impact upon the healing process.Applicants have found that the present compositions and compounds can beused to beneficially regulate and control biologically active proteins,such as growth factor, at the site of a wound. For example, when thepresent compounds and compositions are combined with growth factorsprior to biodelivery as described herein, the compositions and compoundsslowly release this growth factor into the immediate vicinity of thewound, thereby accelerating the wound healing process. It iscontemplated that all growth factors known to accelerate or facilitatewound healing are usable in the present compositions and methods. Growthfactors suitable for this acceleration of wound healing include thoselisted in Table I, as well as brain endothelial cell growth factor andretina-derived growth factor. As described above, heparin binding growthfactors can be used to effect the repair of both soft and hard tissue.The potential uses for interferons, interleukins, and tissue growthfactors are well known in the art.

The invention also relates to methods for the therapeutic administrationof polyanionic saccharide derivatives, or complexes thereof, with aprotein factor, wherein the saccharide derivative is combined with orcomprises a portion of a biocompatible porous solid. The phrase,"biocompatible porous solid" as used herein means a solid which may beapplied or administered to a mammal without provoking a substantialinflammatory response or other substantial adverse effect. Suchbiocompatible porous solids include membranes such as collagen-basedpolymeric membranes, amniotic membranes, and omentum membranes (reviewedin Cobb, 1988, Eur. J. Clin. Investig. 18:321-326). The polyanionicsaccharide derivatives may be immobilized on such membranes in apreferred embodiment by contacting the derivatized saccharide withelectrostatic binding partners on the membrane. Biocompatible poroussolids may also include polymers of ethylene vinyl acetate,methylcellulose, silicone rubber, polyurethane rubber, polyvinylchloride, polymethylacrylate, polyhydroxyethylacrylate, polyethyleneterephthalate, polypropylene, polytetrafluoroethylene, polyethylene,polyfluoroethylene, propylene, cellulose acetate, cellulose andpolyvinyl alcohol (reviewed in Hoffman, Synthetic Polymeric Biomaterialsin Polymeric Materials and Artificial Organs, ACS Symposium Series #256,(G. Gebelein, ed.) 1988). In preferred embodiments, the cyclodextrinstarting materials are co-polymerized with monomers of the biocompatiblepolymer material of the final product composition, so as to create aporous co-polymer. This copolymer is subsequently reacted chemically toprovide the saccharide portion with the anionic substituents required bythis invention. Cyclodextrins can be coupled with reactive groups, suchas amine, amide, carboxylate end groups, etc., contained in thebiocompatible polymer and then subsequently derivatized with ionicsubstituents. More preferably the polysaccharide, such as a cyclodextrinis introduced as a co-reagent in a monomer formulation to be polymerizedto a solid polymer or co-polymer, and the product is contactedsubsequently with suitable agents to derivatize the saccharide componentto add anionic substituents to the degree taught by this invention.Particularly advantageous for such process and products are thosemethods that will produce a polymer or co-polymer example of a flatpolymer product of polyamide polymer, manufactured by 3M Corporation,and used as a bio-compatible patch or dressing on wounds. Thisbiocompatible patch or dressing is designed to physically protect awound from invasion of pathogens, and yet to have sufficient porosity toallow passage of moisture, air, etc. Applicants' invention contemplatesthe coupling of the active polyanionic polysaccharide with a carriercomprising such polymer, or, the coupling of the active anionicsaccharide and a proteinic factor together with a polymeric carrier.Such combination is designed expressly for applications of deliberatepromotion or inhibition of cellular growth processes. The HBGFs bind tothe immobilized, derivatized saccharide-based molecules, eitherincorporated into or already present in biomembranes. Biologicalmembranes such as omentum and amnion are well known in the art as wounddressings. Collagen based synthetic biomembranes are being used in thetreatment of burns. The presence of derivatized saccharide of thepresent invention in natural membranes such as amnion and the ability ofthese derivatives to bind collagen which is used as a base for syntheticmembranes will allow such biomembranes, when combined with thecompositions of the present invention, to be used as novel deliveryvehicles for HBGFs.

A. Restenosis

Arteriosclerosis is a disorder involving thickening and hardening of thewall portions of the larger arteries of mammals, and is largelyresponsible for coronary artery disease, aortic aneurisms and arterialdiseases of the lower extremities. Arteriosclerosis also plays a majorrole in cerebral vascular disease.

Angioplasty has heretofore been a widely used method for treatingarteriosclerosis. For example, percutaneous transluminal coronaryangioplasty (hereinafter "PTCA") was performed over 200,000 times in theUnited States alone during 1988. PTCA procedures involve inserting adeflated balloon catheter through the skin and into the vessel or arterycontaining the stenosis. The catheter is then passed through the lumenof the vessel until it reaches the stenotic region, which ischaracterized by a build up of fatty streaks, fibrous plaques andcomplicated lesions on the vessel wall, which result in a narrowing ofthe vessel and blood flow restriction. In order to overcome the harmfulnarrowing of the artery caused by the arteriosclerotic condition, theballoon is inflated, thus flattening the plaque against the arterialwall and otherwise expanding the arterial lumen.

Although PTCA has produced excellent results and low complication rates,there has, however, been difficulties associated with the use of thistechnique. In particular, the arterial wall being enlarged frequentlyexperiences damage and injury during expansion of the balloon againstthe arterial wall. While this damage itself is not believed to beparticularly harmful to the health or the life of the patient, thehealing response triggered by this damage can cause a reoccurrence ofthe arteriosclerotic condition. In particular, it has been observed thatthe smooth muscle cells associated with the stenotic region of theartery initiate cell division in response to direct or inflammatoryinjury of the artery. As the smooth muscle cells proliferate and migrateinto the internal layer of the artery, they cause thickening of thearterial wall. Initially, this thickening is due to the increased numberof smooth muscle cells. Subsequently, however, further thickening of thearterial wall and narrowing of the lumen is due to increased smoothmuscle cell volume and accumulation of extracellular matrix andconnective tissue. This thickening of the cell wall and narrowing of thelumen following treatment of arteriosclerosis is referred to herein asrestenosis.

Although applicants do not wish to be bound by any theory or theoriesfor the basis of restenosis, it is believed that restenosis is due inpart to the presence of growth factors produced by injured endotheliumwhich activate excessive proliferation of the smooth muscle cells whichare exposed to the endothelial injury. Accordingly, applicants havefound that the present saccharide derivatives, when substantially freeof growth factors prior to biodelivery, are extremely effective forpreventing or at least substantially reducing intimal thickeningfollowing balloon angioplasty. By virtue of their affinity for growthfactors, such compositions can provide an in vivo absorption orreduction of the local concentration and/or diffusion of such growthfactors. That is, such wound site growth factors, whether they areproduced by the cells at the wound site or are otherwise in thebloodstream, can be taken up by the present saccharide derivatives,thereby reducing the restenoic effect of such materials on the woundedtissue.

According to the present methods, mammals, including humans, which havearterial regions subject to angioplasty, are treated by administering tothe mammal a polyanionic saccharide derivative of the present inventionin an amount effective to inhibit arterial smooth muscle cellproliferation. It is contemplated that the degree of restenosisinhibition may vary within the scope hereof, depending upon such factorsas the patient being treated and the extent of arterial injury duringangioplasty. It is generally preferred, however, that the saccharidederivative be administered in an amount effective to cause a substantialreduction in restenosis. As the term is used herein, substantialreduction in restenosis means a post treatment restenosis value of nogreater than about 50%. According to preferred embodiments, the posttreatment restenosis value is no greater than about 25%. As the term isused herein, post-treatment restenosis value refers to the restenosisvalue measured at about one month after angioplasty. The term restenosisvalue refers to the restenosis rate calculated as a loss of greater thanor equal to 50% of the initial gain in minimum lumen diameter achievedby angioplasty.

Thus, the present invention contemplates a method of inhibitingrestenosis in a patient which comprises administering to the patient anamount of a saccharide-based derivative effective to inhibit formationof a restenotic lesion in a patient who has undergone angioplasty. It iscontemplated that the saccharide derivative may be administered before,during and/or after angioplasty treatment of the stenosed artery. Itgenerally preferred that the administration comprise administering thecompound locally at the wound site. In preferred embodiments, localadministration comprises infusing the saccharide derivative directlyinto the injured tissue. In the case of restenosis, such step preferablycomprises infusing the compound directly into the arterial wall at thesite of the angioplasty.

Applicants have surprisingly found that particularly beneficialantirestenoic results are obtained for embodiments in which the step ofadministering the saccharide derivative also comprises the step ofdilating the vessel lumen to effect angioplasty. For example, applicantshave found that a preferred administration step comprises infusing anaqueous suspension or dispersion of saccharide derivative directly intothe arterial wall at the site of balloon angioplasty. This is preferablyaccomplished using a modified infusion balloon catheter having aplurality of holes in the wall of the balloon portion of the catheter.These holes are configured and sized to allow the balloon to be bothinflated and to leak the inflation solution through the wall of theballoon. According to preferred embodiments, the balloon is inflatedunder relatively low pressure conditions, such as 2-3 atmospheres.Examples of porous balloon catheters which may be used to apply thecompositions of the present invention are made by U.S.C.I. Bard andSchneider. Balloons of this type are referred to as Wolinsky balloons or"sweating balloons." It is anticipated that a variety of infusionangioplasty balloon catheters may be used for application of thecompositions of the present invention and that one skilled in the artwould be readily able to determine which types of balloon infusioncatheters would be appropriate. Another technique which involves thelocal administration of the saccharide derivatives of the presentinvention utilizes bioabsorbable intravascular stents. The saccharidesof the present invention, particularly the cyclodextrin polymerderivatives may be incorporated into a bioabsorable stent and that stentpositioned at or near the site of tissue damage.

It will be appreciated by those skilled in the art that the particularcharacteristics and properties of the suspension containing thesaccharide derivative may vary widely depending upon numerous factorsnot necessarily related to the present invention. However, theadministration step preferably comprises infusing an aqueous suspensionor dispersion of polyanionic saccharide derivate particles, andpreferably a suspension of sulfated beta-cyclodextrin polymer particles,ranging in size from about 1 to 600 microns directly into the arterialwall at the site of balloon angioplasty. Applicants believe that suchparticles instilled into the arterial wall will remain present at thesite of application for several days, in sufficient quantity to resultin an inhibition of restenosis.

The aqueous suspension comprises a aqueous carrier of physiologicalsalinity and an active saccharide derivative. The active saccharidederivative is preferably present in an amount ranging from about 1 toabout 30% by weight, and even more preferably from about 5 to about 15%by weight of the composition. In preferred embodiments, derivatizedsaccharides, and preferably cyclodextrin sulfate polymer particles, areapplied at about the time of angioplasty.

In some instances it may be desirable to prevent restenosis but allowangiogenesis. To meet these requirements it is preferred to use adispersion of an Al or Ba salt of a polyanionic saccharide derivative,and even more preferably an Al or Ba salt of a poly sulfatedbeta-cyclodextrin. If it is desired to allow the normal progression ofangiogenesis at the vascular injury site while simultaneously inhibitingrestenosis, it is preferred to use sucralfate, an aluminum salt ofsucrose octasulfate available from the Marian Merrill Dow company,Kansas City, Mo.

B. Inhibition of Intimal Thickening of Vein Grafts

Venous segments are frequently harvested at the time of surgery and usedas bypass grafts to treat vascular occlusive disorders. Specifically,they have been used in the coronary, renal, femoral and poplitealarterial circulations, by way of example. One major limitation of thisform of therapy is that intimal thickening occurs which compromises theluminal cross-sectional area and results in reduced flow. Thisfrequently, but not exclusively occurs at the anastomosis. Applicantspropose that the placement of β-cyclodextrin tetradecasulfate polymericparticles in the perivascular space at the time of surgery, willsubstantially limit the ingrowth of smooth muscle cells into the intimaand will improve the long term success of these grafts.

C. Angiogenesis

Angiogenesis is the formation of new blood vessels. Angiogenic stimulicause the elongation and proliferation of endothelial cells and thegeneration of new blood vessels. A number of the HBGFs are known topromote angiogenesis. The new blood vessels produced by angiogenesisresult in neovascularization of tissue.

There are a variety of diseases associated with deficient blood supplyto tissue and organs. A deficiency of this kind, known as ischaemia, maybe due to the functional constriction or actual obstruction of a bloodvessel. These diseases can be grouped into cardiac, cerebral andperipheral ischemic diseases. Cardiac ischaemia may result in chronicangina or acute myocardial infarction. Cerebral ischaemia may result ina stroke. Peripheral ischaemia may result in a number of diseasesincluding arterial embolism and frostbite. In severe cases of peripheralischaemia, necrosis of the tissues supplied by the occluded bloodvessels necessitates amputatior. To overcome ischaemia, an alternativeblood supply to the affected tissue must be established.

According to preferred embodiments, angiogenesis is promoted by firstcontacting a saccharide derivative of the present invention with growthfactor(s) and then administering the composition locally to the locationof the ischemic tissue, by hypodermic injection for example, to promoteangiogenesis and the formation of collateral blood vessels. As the termis used herein, collateral blood vessels are blood vessels which areabsent under normal physiological conditions but develop in response toappropriate stimuli, such as the presence of HBGFs. It is anticipatedthat administration of compositions which include saccharide derivativeand growth factor will result in the formation of collateral bloodvessels and revascularization of ischemic tissue.

In preferred embodiments, angiogenesis is promoted by methods in whichthe saccharide derivative comprises a highly anionic cyclodextrinderivative or a salt form of same, and even more preferably apolysulfated polymer or copolymer of a cyclodextrin. It is preferredthat the cyclodextrin derivative be combined with basic fibroblastgrowth factor at a cyclodextrin:basic fibroblast growth factor weightratio of from about 10:1 to 100:1.

D. Tissue and Organ Grafts or Transplants

As described above, HBGFs are known to stimulate neovascularization andendothelial cell growth. In transplantation, the graft represents awound, and success of the grafting procedure depends critically on therapidity of establishing an adequate blood supply to the grafted ortransplanted tissue. Thus, we envision the application of thecompositions of the present invention combined with growth factor(s) atthe site of the graft to promote the establishment of an adequate bloodsupply to the grafted or transplanted tissue. The growthfactor-containing compositions may be coated on the surfaces to bejoined, sprayed on the surfaces, or applied in the form of an aqueoussuspension with or without viscosity enhancers such as glycerol. Inaddition, the organ or tissue to be grafted or transplanted may bepresoaked in a treating solution containing the compositions of thepresent invention, prior to transplantation. The compositions of thepresent invention may also be injected into the transplant site orsurface of both items to be joined.

In a preferred method for preparing the compositions used in treatinggrafted or transplanted tissue and organs, the saccharide derivatives ofthe present invention are precontacted with growth factor containingorganic sources (e.g., tissue or organ debris, ground matter, or liquidextract) so as to extract the growth factors present in these sources.In highly preferred methods, the organic source used for contact isabout 10 to about 100 times greater in volume than the transplanted orgrafted tissue to be treated by the composition. A more direct and oftenmore economic method will involve contacting the saccharide derivativesof the present invention with growth factor substances created byrecombinant biochemical and biotechnological procedures. In this mannerspecific growth factor proteins are more readily chosen for acontemplated therapeutic application.

E. Bone Grafting and Transplantation

The response of bone to injuries such as fractures, infection andinterruption of blood supply is relatively limited. In order for thedamaged bone tissue to heal, dead bone must be resorbed and new bonemust be formed, a process carried out in association with new bloodvessels growing into the involved area. HBGFs can induceneovascularization and the proliferation of bone forming cells. It istherefore contemplated to use the present compounds in combination withgrowth factor for the purposes of aiding the healing of bone fractures,the joining of implanted and host bone, and the mineralization of bone(where such is intended).

In preferred embodiments, the present saccharide derivatives arecombined with growth factors and powdered bone substance and/or finelydispersed demineralized bone matter to form a paste. Suitable methodsfor preparation of such a paste are presented in Repair of MajorCranio-Orbital Defects with an Elastomer Coated Mesh and Autogenous BonePaste, Mutaz B. Habal et al., 61:3, Plastic and Reconstructive Surgery,394, 396 (1978). The bone tissue used to produce the paste may beobtained from iliac crest or calvarium. It is preferred to useautogenous bone for implant purposes and to use partially demineralizedbone over fully demineralized bone powder. Demineralized bone powderobtained from allogenic and xenogeneic sources may be used in preparingthe bone powder. To make a soft paste absorbable cellulose cotton orsimilar material may be used. Although applicants do not wish to bebound by any theory or theories, it is thought that the bone pasteproduced by these methods functions as an induction matrix from whichnew bone will form after being invaded with a network of blood vessels.The paste is applied to the surfaces of bone to be joined in implantprocedures or used to fill fractures of contour bone to be repaired.

F. Skin Ulcer Healing

One debilitating disorder affecting millions of people including, butlimited to the aged, paraplegics, trauma victims, and diabetics arecutaneous nonhealing skin ulcers or decubiti. In many cases, inadequateblood supply to the damaged tissue prevents the delivery of adequatenutrients for healing. It is anticipated that the application ofpolymeric beads of cyclodextrin derivatives, preabsorbed withcombinations of compounds such as epidermal growth factor and basicfibroblast growth factor, to the ulcer directly, will lead to increasedangiogenesis, improved blood supply, increased keratinocyte ingrowth,and faster ulcer closure and healing.

G. Dermatological Applications

The control of blood vessel growth is an important aspect of normal andof pathological states encountered in dermatology. In particular, theabnormal growth of cellular materials and vessels accompanies severalpathological sates, psoriasis being one prominent example. In many casesexcesses of growth stimulating protein factors are involved.Abnormalities of this type are often associated with imbalances inproteinic growth factors.

For example, in the case of patients with cutaneous mastocytosis,extracts from involved skin had 15-fold higher levels of chymotrypticactivity than extracts of uninvolved skin or from control samples ofpatients without such deficiency. (See Human Skin Chymotryptic Protease,N. M. Schechter, J. E. Fraki, J. C Geesin, G. S. Lazarus, J. Biol.Chem., 258, 2973-2978, 1983. The Chymase Involved Is a Heparin BindingFactor (See S. Sayama, R. V. Iozzo, G. S. Lazarus, N. M. Schechter,Human Skin Chymotrypsin-like Proteinase Chymase, J. Biol. Chem. 262,6808-6815, 1987. It appears that the chymotrypsin like proteases candegrade the epidermal junction and can result in epidermal-dermalseparation (See Sayama et al. above).

Another example of a growth promoting factor involved in dermalabnormalcies is epidermal plasminogen activator, which is elevated in avariety of dermal pathologies (See Epidermal Plasminogen Activator isAbnormal in Cutaneous Lesions', P. J. Jensen et al., J. Invest. Dermat.90-777-782, 1988).

Certain embodiments of this invention, namely highly sulfated soliddispersions or other physical variants of highly sulfatedpolysaccharides, and preferably those comprising cyclodextrinstructures, are particularly amenable to dermal therapy in those caseswhere excess growth of cellular components is involved. In such case theagents of the present invention can be introduced at or near the tissueinvolved. This may be accomplished by cutaneous or sub-cutaneousinjection of fine particle dispersion of the agent, or the implantationof solid polymer shapes suitably shaped for effective contact, or theagent may be comprised in material such as patches, or other suitableforms of externally applied materials containing agents of theinvention.

It will be understood that depending on the pathology and diseasecondition, the application of the agents of this invention withoutpre-contacting with proteinic growth factor is contemplated. This willbe the case in conditions as exemplified above, where it is intended toreduce any growth promoting factor or factors.

In other cases of dermal damage or disease, and in certain phases oftreatment, it may be desirable to use the combined proteinic factors.This would be the case in connection with healing processes whereangiogenesis, that is the establishment of new and added blood suppliesare desired.

EXAMPLES

The following examples are provided to illustrate this invention.However, they are not to be construed as necessarily limiting the scopeof the invention, which scope is determined by the appended claims. Allamounts and proportions shown are by weight unless explicitly stated tobe otherwise.

Example 1 Preparation of Sulfated Beta-Cyclodectrin Polymer

Beta-cyclodextrin polymer beads (American Maize Products) of 20-60 meshparticle size were derivatized to form a novel immobilized CD polymersulfate derivative according to the present invention. The compositionapproaches a degree of sulfation of nearly two sulfates per glucose ringof the CD polymer. About 0.4 g of carefully dried polymer were reactedwith about 1.7 g of 6 trimethylammonium sulfur trioxide complex(Aldrich) in about 100 ml of dried dimethylformamide (DMF), with mildagitation at about 62 to 72° C. for 3 to 4 days. The solids were washedin DMF, reacted with 30% aqueous sodium acetate for 24 hours, and washedand stored in distilled water. The sulfur content of the product wasabout 14.7 wt. %. This compares favorably to the value of 17.5% if thepolymer mass were composed 100% of β-cyclodextrin tetradecasulfatewithout cross-linking components, and all glucose hydroxyl units weresterically available (which cannot be expected for the polymer).

Example 2 Preparation of Derivatives of Cyclodextrin

(A) β CD-TDS(Na):

β-cyclodextrin (99% pure dihydrate) was purchased from Chemalog (adivision of General Dynamics Corp.), South Plainfield, N.J.

About 5.0 grams of β-cyclodextrin (about 4.4 mmoles, i.e., about 92 meq)--OH) was dissolved in about 250 ml of dimethyl-formamide (DMF). To thissolution was added about 15 grams of (CH₃)₃ N--SO₃ (about 108 mmoles) ina single portion and the reaction mixture was heated to about 70° C.After two hours at about 70° C., a gummy material began to precipitate.The reaction mixture was maintained at 70° C. with vigorous stirring,and then cooled to room temperature. The DMF layer was then decanted anddiscarded, and the solid residue was dissolved in about 250 ml of waterfollowed by addition of about 75 ml of 30% sodium acetate. The mixturewas stirred vigorously for 4 hours and then poured into about 4000 ml ofethanol. After standing overnight, the mixture was filtered to recoverthe crystallized solids. The filter cake was washed with ethanol(absolute) followed by diethyl ether. The product was then dried undervacuum over P₂ O₅. About 10.3 grams of white powder was recovered. Theproduct was hygroscopic.

The product was analyzed under conditions such that sorption of waterwas minimized. Elemental analysis gave the following results: C=18.84,H=2.65, S=17.33 (Calculated for C₆ H₈ O₁₁ S₂ Na₂ ; C=19.67, H=2.19,S=17.49). α!D²² =75° (C=2.63 in 0.5M NaCl). The analysis corresponds tothat expected for an average substitution of two hydroxyl groups foreach glucopyranose unit, i.e., 14 hydroxyls per CD molecules. Thecalculated yield for such β-CD-TDS salt is 10.96 grams, about 6% higherthan the observed 10.3 grams.

(B) α- and γ-CD-S (Na salt):

The procedure described above was used for these preparations exceptthat about 86 mmoles of CH₃ N--SO₃ was used with β-CD, and about 117mmoles with the γ-CD.

The sulfated α-CD salt analyzed C=18.76; H=2.60; S=16.22. Thiscorresponds on average to a substitution of about 11.7 hydroxyl unitsper β-CD molecule.

The sulfated γ-CD salt analyzed C=18.92; H=2.69; S=14.84. Thiscorresponds on average to a substitution of about 14 hydroxyl groups perγ-CD molecule.

(C) β CD-SO₄ (Na salt) (7.1 wt %) S):

About 1.0 gm of β-cyclodextrin was dissolved into about 50 ml of DMF. Tothis solution was added about 883 mg of (CH₃ N SO₃ (7.2 equivalents).The solution was held at about 75° C. for about 12 hours, at which timeno precipitate had formed. The reaction mixture was cooled to roomtemperature. To the solution was added about 200 ml of ethanol. Theresulting colloidal solution was then poured into about 600 ml ofdiethyl ether. A white solid formed in 2 hours. The solid was collectedby filtration and then was dissolved in about 30 ml H₂ O. This solutionwas stirred for 2 hours. After stirring, the solution was poured intoabout 900 ml of 2:1 EtOH-Et₂ O solution. Crystals formed over an 8 hourperiod. The crystals were collected and washed with Et₂ O. The productwas dried over P₂ O₅ under vacuum. About 1.18 gm of powder wasrecovered. (72.4% yield).

Elemental analysis of the product showed C=32.49; H=4.99; and S=7.06.This corresponds on average to a substitution of about 3.5 hydroxyls perβ-CD molecule.

(D) β-CD-Propoxylate˜14 SO₄

β-CD-(hydroxy-n-propyl ether) was obtained from American Maize-ProductsCo. (Hammond, Ind.) and the procedure described above was used toprepare the sulfate salt, β-CD-(˜4Pr˜14 SO₄).

Example 3 Preparation of Growth Factors

Human recombinant basic fibroblast growth factor (bFGF) was provided byTakeda Chemical Industries, Ltd. It was purified from E. coli aspreviously described (Kurokawa et al., 1987, FEBS. Letters 213:189-194and Iwane et al., 1987, Biochem. Biophys. Res. Commun. 146:470-477).

Rat chondrosarcoma-derived growth factor (ChDGF) was isolated from thetransplantable tumor as previously described (Shing et al., 1984,Science 223:1296-1298). About one hundred ml of the crude extractprepared by collagenase digestion of the tumor was diluted (1:1) withabout 0.6M NaCl in about 10 mM Tris, pH 7 and loaded directed onto aheparin-Sepharose® column (1.5×9 cm) pre-equilibrated with the samebuffer. The column was rinsed with about 100 ml of about 0.6M NaCl inabout 10 mM Tris, pH 7. ChDGF was subsequently eluted with about 18 mlof about 2M NaCl in about 10 mM Tris, pH 7.

Example 4 Beta-Cyclodextrin Affinity Chromatography of FGF

The insoluble sulfated beta-cyclodextrin polymer (about 0.5 ml bedvolume), was incubated with about 0.5 ml of about 0.1M NaCl, about 10 mMTris, about pH 7 containing about 1,000 units of human recombinant bFGFat about 4° C. for about 1 hour with mixing. Subsequently, the polymerwas rinsed stepwise with about 2 ml each of about 0.1, 0.6, and 2M NaClin about 10 mM Tris, pH 7. All fractions eluted from the polymer wereassayed for growth factor activity.

Example 5 Growth Factor Assay

Growth factor activity was assessed by measuring the incorporation of ³H!thymidine into the DNA of quiescent, confluent monolayers of BALB/cmouse 3T3 cells in 96-well plates. One unit of activity was defined asthe amount of growth factor required to stimulate half-maximal DNAsynthesis in 3T3 cells (about 10,000 cells/0.25 ml of growthmedium/well). For determination of specific activities, proteinconcentrations of the crude extract and the active fraction eluted fromheparin-Sepharose column were determined by the method of Lowry et al.(1952, J. Biol. Chem. 193:265-275). Protein concentrations of the puregrowth factor were estimated by comparing the intensities ofsilver-stained polypeptide bands of SDS-polyacrylamide gel to those ofthe molecular weight markers.

Example 6 Affinity of Fibroblast Growth Factor for Beta-CyclodextrinTetradecasulfate Polymer

Human recombinant bFGF (about 1000 units) was incubated with sulfatedbeta-cyclodextrin polymer. The polymer was subsequently eluted stepwisewith about 0.1M, 0.6M, and 2M NaCl. The results are shown in FIG. 3.

While most of the growth factor activity remained bound to the polymerat about 0.6M NaCl, about 230 units of the activity was recovered wheneluted with about 2M NaCl. These results indicate that basic fibroblastgrowth factor has a very strong affinity for beta-cyclodextrintetradecasulfate and is at least comparable to that of FGF for heparin.The activity peak was analyzed by SDS polyacrylamide gel electrophoresisfollowed by a silver stain. Lane 2 in FIG. 4 shows the polypeptide bandof basic fibroblast growth factor.

The affinities of heparin and beta-cyclodextrin tetradecasulfate forchondrosarcoma derived growth factor were also tested. Chondrosarcomaextracts which contained about 500 units of growth factor activity wereincubated individually with heparin-Sepharose® and beta-cyclodextrintetradecasulfate polymer. The beads wore subsequently eluted stepwisewith about 0.1M, 0.6M, and about 2M NaCl. The results are shown in FIG.5. Approximately 32% and 68% of the total activity was recovered at 2MNaCl with heparin Sepharose® and beta-cyclodextrin tetradecasulfatepolymer, respectively.

What is claimed is:
 1. A method of promoting angiogenesis in a tissue ofa mammal comprising administering locally to the tissue a compositioncomprising a growth factor combined with a polyanionic cyclodextrinderivative in a physiologically acceptable carrier in an amounteffective to promote angiogenesis, the cyclodextrin derivativecomprising at least one cyclodextrin monomer and having a bodytemperature solubility of less than about 15 grams/100 ml of distilledwater.
 2. The method of claim 1, wherein at least a portion of thecyclodextrin derivative is a solid particulate dispersed or suspended inthe carrier.
 3. The method of claim 1, wherein the cyclodextrinderivative has a body temperature solubility of less than about 1gram/100 ml of distilled water.
 4. The method of claim 3, wherein thecyclodextrin derivative has a body temperature solubility of less thanabout 1 milligram/100 ml of distilled water.
 5. The method of claim 4,wherein the cyclodextrin derivative is substantially insoluble in waterat body temperature.
 6. The method of claim 1, wherein each cyclodextrinmonomer comprises at least 6 sugar units, wherein each sugar unit has anaverage of about 1 to about 4 substituents, and wherein each substituentis selected from the group consisting of sulfate, sulfonate, andphosphate.
 7. The method of claim 6, wherein each sugar unit has anaverage of about 1.0 to about 1.4 substituents.
 8. The method of claim6, wherein each cyclodextrin monomer further comprises at least onenonanionic group selected from the group consisting of alkyl, aryl,ester, ether, thioester, thioether, and --COOH.
 9. The method of claim1, wherein at least one cyclodextrin monomer comprises a salt ofpolyanionic alpha-, beta-, or gamma-cyclodextrin.
 10. The method ofclaim 1, wherein the local administration comprises contacting thetissue with a biocompatible porous solid which comprises thecyclodextrin derivative.
 11. The method of claim 1, wherein the growthfactor is a heparin-binding growth factor.
 12. The method of claim 1,wherein the growth factor is selected from the group comprising basicfibroblast growth factor and epidermal growth factor.
 13. The method ofclaim 12, wherein the growth factor is basic fibroblast growth factorand wherein the cyclodextrin derivative:basic fibroblast growth factorweight:weight ratio is about 10:1 to about 100:1.
 14. The method ofclaim 12, wherein the tissue is a skin ulcer, wherein the growth factoris epidermal growth factor and wherein at least a portion of thecyclodextrin derivative is a solid particulate dispersed or suspended inthe carrier.
 15. The method of claim 1, wherein the local administrationcomprises delivering the composition to the tissue by hypodermicinjection.
 16. The method of claim 1, wherein the cyclodextrinderivative comprises a derivatized cyclodextrin polymer.
 17. A method ofpromoting wound healing in a tissue of a mammal comprising administeringlocally to the tissue a growth factor combined with a polyanioniccyclodextrin derivative in a physiologically acceptable carrier in anamount effective to promote wound healing, the cyclodextrin derivativecomprising at least one cyclodextrin monomer and having a bodytemperature solubility of less than about 15 grams/100 ml of distilledwater.
 18. The method of claim 17, wherein the growth factor is selectedfrom the group consisting of brain endothelial cell growth factor,retina-derived growth factor, interleukin-1, interleukin-2, interferonalpha, interferon gamma, tumor necrosis factor alpha, epidermal growthfactor, acidic fibroblast growth factor, basic fibroblast growth factor,insulin-like growth factor-1, insulin-like growth factor-2,platelet-derived growth factor, transforming growth factor-alpha,transforming growth factor-beta, and a heparin-binding growth factor.19. The method of claim 17, wherein at least a portion of thecyclodextrin derivative is a solid particulate dispersed or suspended inthe carrier.
 20. The method of claim 17, wherein the cyclodextrinderivative has a body temperature solubility of less than about 1gram/100 ml of distilled water.
 21. The method of claim 19, wherein thecyclodextrin derivative has a body temperature solubility of less thanabout 1 milligram/100 ml of distilled water.
 22. The method of claim 21,wherein the cyclodextrin derivative is substantially insoluble in waterat body temperature.
 23. The method of claim 17, wherein eachcyclodextrin monomer comprises at least 6 sugar units, wherein eachsugar unit has an average of about 1 to about 4 substituents, andwherein each substituent is selected from the group consisting ofsulfate, sulfonate, and phosphate.
 24. The method of claim 23, whereineach sugar unit has an average of about 1.0 to about 1.4 substituents.25. The method of claim 23, wherein each cyclodextrin monomer furthercomprises at least one nonanionic group selected from the groupconsisting of alkyl, aryl, ester, ether, thioester, thioether, and--COOH.
 26. The method of claim 17, wherein at least one cyclodextrinmonomer comprises a salt of polyanionic alpha-, beta-, orgamma-cyclodextrin.
 27. The method of claim 17, wherein the localadministration comprises contacting the tissue with a biocompatibleporous solid which comprises the cyclodextrin derivative.
 28. The methodof claim 17, wherein the cyclodextrin derivative comprises a derivatizedcyclodextrin polymer.
 29. A method of promoting establishment of bloodsupply to a tissue which is grafted or transplanted into a mammal, themethod comprising administering locally to the tissue a compositioncomprising a growth factor combined with a polyanionic cyclodextrinderivative in a physiologically acceptable carrier in an amounteffective to promote establishment of blood supply, the cyclodextrinderivative comprising at least one cyclodextrin monomer and having abody temperature solubility of less than about 15 grams/100 ml ofdistilled water.
 30. The method of claim 29, wherein the growth factoris selected from the group consisting of brain endothelial cell growthfactor, retina-derived growth factor, interleukin-1, interleukin-2,interferon alpha, interferon gamma, tumor necrosis factor alpha,epidermal growth factor, acidic fibroblast growth factor, basicfibroblast growth factor, insulin-like growth factor-1, insulin-likegrowth factor-2, platelet-derived growth factor, transforming growthfactor-alpha, transforming growth factor-beta, and a heparin-bindinggrowth factor.
 31. The method of claim 29, wherein at least a portion ofthe cyclodextrin derivative is a solid particulate dispersed orsuspended in the carrier.
 32. The method of claim 29, wherein thecyclodextrin derivative has a body temperature solubility of less thanabout 1 gram/100 ml of distilled water.
 33. The method of claim 32,wherein the cyclodextrin derivative has a body temperature solubility ofless than about 1 milligram/100 ml of distilled water.
 34. The method ofclaim 33, wherein the cyclodextrin derivative is substantially insolublein water at body temperature.
 35. The method of claim 29, wherein eachcyclodextrin monomer comprises at least 6 sugar units, wherein eachsugar unit has an average of about 1 to about 4 substituents, andwherein each substituent is selected from the group consisting ofsulfate, sulfonate, and phosphate.
 36. The method of claim 35, whereineach sugar unit has an average of about 1.0 to about 1.4 substituents.37. The method of claim 35, wherein each cyclodextrin monomer furthercomprises at least one nonanionic group selected from the groupconsisting of alkyl, aryl, ester, ether, thioester, thioether, and--COOH.
 38. The method of claim 29, wherein at least one cyclodextrinmonomer comprises a salt of polyanionic alpha-, beta-, orgamma-cyclodextrin.
 39. The method of claim 29, wherein the localadministration comprises contacting the tissue with a biocompatibleporous solid which comprises the cyclodextrin derivative.
 40. The methodof claim 29, wherein the composition is administered to the tissue priorto transplanting or grafting the tissue into the mammal.
 41. The methodof claim 29, wherein the composition is administered to the tissueconcurrently with transplanting or grafting the tissue into the mammal.42. The method of claim 29, wherein the composition is administered tothe tissue following transplanting or grafting the tissue into themammal.
 43. The method of claim 29, wherein the cyclodextrin derivativecomprises a derivatized cyclodextrin polymer.
 44. A method of promotingmineralization of bone tissue of a mammal comprising administeringlocally to the bone tissue a composition comprising a growth factorcombined with a polyanionic cyclodextrin derivative in a physiologicallyacceptable carrier in an amount effective to promote mineralization, thecyclodextrin derivative comprising at least one cyclodextrin monomer andhaving a body temperature solubility of less than about 15 grams/100 mlof distilled water.
 45. The method of claim 44, wherein the growthfactor is selected from the group consisting of interleukin-1,interleukin-2, interferon alpha, interferon gamma, tumor necrosis factoralpha, epidermal growth factor, acidic fibroblast growth factor, basicfibroblast growth factor, insulin-like growth factor-1, insulin-likegrowth factor-2, platelet-derived growth factor, transforming growthfactor-alpha, transforming growth factor-beta, and a heparin-bindinggrowth factor.
 46. The method of claim 44, wherein at least a portion ofthe cyclodextrin derivative is a solid particulate dispersed orsuspended in the carrier.
 47. The method of claim 44, wherein thecyclodextrin derivative has a body temperature solubility of less thanabout 1 gram/100 ml of distilled water.
 48. The method of claim 47,wherein the cyclodextrin derivative has a body temperature solubility ofless than about 1 milligram/100 ml of distilled water.
 49. The method ofclaim 48, wherein the cyclodextrin derivative is substantially insolublein water at body temperature.
 50. The method of claim 44, wherein eachcyclodextrin monomer comprises at least 6 sugar units, wherein eachsugar unit has an average of about 1 to about 4 substituents, andwherein each substituent is selected from the group consisting ofsulfate, sulfonate, and phosphate.
 51. The method of claim 50, whereineach sugar unit has an average of about 1.0 to about 1.4 substituents.52. The method of claim 50, wherein each cyclodextrin monomer furthercomprises at least one nonanionic group selected from the groupconsisting of alkyl, aryl, ester, ether, thioester, thioether, and--COOH.
 53. The method of claim 44, wherein at least one cyclodextrinmonomer comprises a salt of polyanionic alpha-, beta-orgamma-cyclodextrin.
 54. The method of claim 44, wherein the localadministration comprises contacting the bone tissue with a biocompatibleporous solid which comprises the cyclodextrin derivative.
 55. The methodof claim 44, wherein the composition further comprises a demineralizedbone matter composition.
 56. The method of claim 55, wherein thedemineralized bone matter composition is selected from the groupconsisting of partially demineralized autogenous bone obtained from theiliac crest, partially demineralized autogenous bone obtained from thecalvarium, fully demineralized autogenous bone, partially demineralizedallogenic bone obtained from the iliac crest, partially demineralizedallogenic bone obtained from the calvarium, fully denmineralizedallogenic bone, partially demineralized xenogeneic bone obtained fromthe iliac crest, partially demineralized xenogeneic bone obtained fromthe calvarium, and fully demineralized xenogeneic bone.
 57. The methodof claim 44, wherein the local administration comprises applying thecomposition to the surface of the bone tissue.
 58. The method of claim44, wherein the local administration comprises filling a void within thebone tissue with the composition.
 59. The method of claim 44 wherein thecyclodextrin derivative comprises a derivatized cyclodextrin polymer.60. A method of modulating proliferation of an endothelial cell in amammal, the method comprising administering locally to the endothelialcell a composition comprising a polyanionic cyclodextrin derivative anda physiologically acceptable carrier in an amount effective to modulateproliferation of the endothelial cell, the cyclodextrin derivativecomprising at least one cyclodextrin monomer and having a bodytemperature solubility of less than about 15 grams/100 ml of distilledwater.
 61. The method of claim 60, wherein modulating proliferation ofan endothelial cell comprises promoting proliferation of the endothelialcell, and wherein the composition frrther comprises a growth factorselected from the group consisting of interleukin-1, interleukin-2,interferon alpha, interferon gamma, tumor necrosis factor alpha,epidermal growth factor, acidic fibroblast growth factor, basicfibroblast growth factor, insulin-like growth factor-1, insulin-likegrowth factor-2, platelet-derived growth factor, transforming growthfactor-alpha, transforming growth factor-beta, and a heparin-bindinggrowth factor.
 62. The method of claim 60, wherein modulatingproliferation of an endothelial cell comprises inhibiting proliferationof the endothelial cell.
 63. The method of claim 60, wherein at least aportion of the cyclodextrin derivative is a solid particulate dispersedor suspended in the carrier.
 64. The method of claim 60, wherein thecyclodextrin derivative has a body temperature solubility of less thanabout 1 gram/100 ml of distilled water.
 65. The method of claim 64,wherein the cyclodextrin derivative has a body temperature solubility ofless than about 1 milligram/100 ml of distilled water.
 66. The method ofclaim 60, wherein the cyclodextrin derivative is substantially insolublein distilled water at body temperature.
 67. The method of claim 60,wherein each cyclodextrin monomer comprises at least 6 sugar units,wherein each sugar unit has an average of about 1 to about 4substituents, and wherein each substituent is selected from the groupconsisting of sulfate, sulfonate, and phosphate.
 68. The method of claim67, wherein each sugar unit has an average of about 1.0 to about 1.4substituents.
 69. The method of claim 67, wherein each cyclodextrinmonomer further comprises at least one nonanionic group selected fromthe group consisting of alkyl, aryl, ester, ether, thioester, thioether,and --COOH.
 70. The method of claim 60, wherein at least onecyclodextrin monomer comprises a salt of polyanionic alpha-, beta-, orgamma-cyclodextrin.
 71. The method of claim 60, wherein the localadministration comprises contacting the dermal tissue with abiocompatible porous solid which comprises the cyclodextrin derivative.72. The method of claim 60, wherein the local administration is selectedfrom the group consisting of cutaneous injection, subcutaneousinjection, implantation, application of a patch comprising thecomposition, and topical application.
 73. The method of claim 60,wherein the local administration is selected from the group consistingof cutaneous injection, subcutaneous injection, implantation, andtopical application.
 74. The method of claim 60, wherein thecyclodextrin derivative comprises a derivatized cyclodextrin polymer.75. The method of claim 61, wherein the cyclodextrin derivativecomprises a derivatized cyclodextrin polymer.
 76. The method of claim60, wherein the endothelial cell is a component of an endothelialtissue.
 77. The method of claim 76, wherein the endothelial tissue isthe luminal surface of a blood vessel.
 78. The method of claim 56,wherein the local administration comprises administering the compound tothe endothelial cell using a balloon angioplasty catheter.
 79. Themethod of claim 62, wherein the endothelial cell is a vascularendothelial cell.
 80. The method of claim 79, wherein inhibitingproliferation of the vascular endothelial cell comprises inhibitingangiogenesis.